Reduction In Bacterial Colonization By Administering Bacteriophage Compositions

ABSTRACT

The present invention provides a method for reducing the risk of bacterial infection or sepsis in a susceptible patient by treating the susceptible patient with a pharmaceutical composition containing bacteriophage of one or more strains which produce lytic infections in pathogenic bacteria. Preferably, treatment of the patient reduces the level of colonization with pathogenic bacteria susceptible to the bacteriophage by at least one log. In a typical embodiment, the susceptible patient is an immunocompromised patient selected from the group consisting of leukemia patients, lymphoma patients, carcinoma patients, sarcoma patients, allogeneic transplant patients, congenital or acquired immunodeficiency patients, cystic fibrosis patients, and AIDS patients. In a preferred mode, the patients treated by this method are colonized with the pathogenic bacteria subject to infection by said bacteriophage.

RELATED APPLICATIONS

The present application is related to U.S. Provisional PatentApplication Nos. 60/175,415 and 60/175,416, filed Jan. 11, 2000, and60/205,240, filed May 19, 2000. In addition, the present application isrelated to U.S. Provisional Patent Application No. 60/175,377 filed Jan.11, 2000. The disclosures of these provisional applications areincorporated herein, by reference, in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to the field of medical treatment andprevention of infections diseases; in particular, use of therapeuticcompositions containing bacteriophage to reduce or eliminatecolonization with potentially pathogenic bacteria (including bacterialstrains resistant to many or most commonly used antimicrobial agents),thereby reducing the risk of subsequent disease occurrence.

2. Description of Related Art

Vancomycin-Resistant Enterococcus

Over the last ten years there has been an emergence of bacterialpathogens, which demonstrate resistance to many, if not allantimicrobial agents. This is particularly relevant in the institutionalenvironment where nosocomial pathogens are under selective pressure dueto extensive antimicrobial usage. A particular problem in this regardhas been vancomycin-resistant enterococci (VRE), which are not treatablewith standard classes of antibiotics. Despite the recent release of twodrugs to which VRE are susceptible (quinupristin/dalfopristin andlinezolid [Plouffe J F, Emerging therapies for serious gram-positivebacterial infections: A focus on linezolid. Clin Infect dis 2000 Suppl4:S144-9), these microorganisms remain an important cause of morbidityand mortality in immunocompromised patients.

Enterococci are gram positive facultatively anaerobic cocci found in avariety of environmental sources including soil, food and water. Theyare also a common colonizing bacterial species in the human intestinaltract (i.e., the intestinal tract serves as a reservoir for themicroorganism). Although the taxonomy of enterococci has not beenfinalized, it is generally accepted that the genus consists of 19species.

Antibiotic management of serious enterococcal infections has always beendifficult due to the intrinsic resistance of the organisms to mostantimicrobial agents [(Arden, R. C, and B. E. Murray, 1994,“Enterococcus: Antimicrobial resistance.” In: Principles and Practice ofInfectious Diseases Update, volume 2, number 4 (February, 1994). NewYork: Churchill Livingstone, Inc. 15 pps; Landman, D., and J. M. Quale,1997, “Management of infections due to resistant enterococci: a reviewof therapeutic options.” J. Antimicrob. Chemother., 40:161-70;Moellering, R. C., 1998, “Vancomcyin-resistant enterococci.” Clin.Infect. Dis. 26:1196-9]. In the 1970's enterococcal infections weretreated with the synergistic combination of a cell wall active agentsuch as penicillin and are aminoglycoside (Moellering, at al. (1971),“Synergy of penicillin and gentamicin against enterococci.” J Infect.Dis., 124:S207-9; Standiford, at al. (1970), “Antibiotic synergism ofenterococci: relation to inhibitory concentrations.” Arch. Intern. Med.,126: 255-9). However, during the 1980's enterococcal strains with highlevels of aminoglycoside resistance and resistance to penicillin,mediated both by a plasmid-encoded β-lactamase and by changes inpenicillin binding proteins, appeared (Mederski-Samoraj, et al. (1983),“High level resistance to gentamicin in clinical isolates ofenterococci.” J. Infect. Dis., 147:751-7; Uttley, et al. (1988),“Vancomycin resistant enterococci.” Lancet i:57-8). In 1988 the firstVRE isolates were identified (Leclercq, at al. (1988), “Plasmid mediatedresistance to vancomycin and teicoplanin in Enterococcus faecium.” NEngl. J. Med., 319:157-61). Such organisms, called VRE because ofresistance to vancomycin, are also resistant to thepenicillin-aminoglyroside combination. VRE includes strains of severaldifferent enterococcal species with clinically significant VREinfections caused by Enterococcus faecium and Enterococcus faecalis.

Enterococci can cause a variety of infections including wound infection,endocarditis, urinary tract infection and bacteremia. AfterStaphylococcus aureus and coagulase negative staphylococci, enterococciare the most common cause of nosocomial bacteremia. Amongimmunocompromised patients, intestinal colonization with VRE frequentlyprecedes, and serves as a risk factor for, subsequent VRE bacteremia(Edmond, et al. (1995), “Vancomycin resistant Enterococcus faeciumbacteremia: Risk factors for infection.” Clin. Inf. Dis., 20:1126-33;Tornieporth, N. G., R. B. Roberts, J. John, A. Hefner, and L. W. Riley,1996, “Risk factors associated with vancomycin-resistant Enterococcusfaecium infection or colonization in 145 matched case patients andcontrol patients.” Clin. Infect. Dis., 23:767-72.]. By using pulse fieldgel electrophoresis as a molecular typing tool investigators at theUniversity of Maryland at Baltimore and the Baltimore VA Medical Centerhave shown VRE strains causing bacteremia in cancer patients are almostalways identical to those which colonize the patients gastrointestinaltract (Roghmann M C, Qaiyumi S, Johnson J A, Schwalbe R, Morris J G(1997), “Recurrent vancomycin-resistant Enterococcus faecium bacteremiain a leukemia patient who was persistently colonized withvancomycin-resistant enterococci for two years.” Clin Infect Dis24:514-5). The risk of acquiring VRE increases significantly when thereis a high rate of VRE colonization among patients on a hospital ward orunit (i.e., when there is high “colonization pressure”). In one study inthe Netherlands, colonization pressure was the most important variableaffecting acquisition of VRE among patients in an intensive care unit(Bonten M J, et al, “The role of “colonization pressure” in the spreadof vancomycin-resistant enterococci: an important infection controlvariable.” Arch Intern Med 1998; 25:1127-32). Use of antibiotics hasbeen clearly shown to increase the density, or level of colonization, inan individual patient (Donskey C J et al, “Effects of antibiotic therapyon the density of vancomycin-resistant enterococci in the stool ofcolonized patients.” N Engl J Med 2000; 343:1925-32): this, in turn,would appear to increase the risk of subsequent infection, and the riskof transmission of the organism to other patients.

Multi-Drug Resistant Staphylococcus aureus (MDRSA)

S. aureus is responsible for a variety of diseases ranging from minorskin infections to life-threatening systemic infections, includingendocarditis and sepsis [Lowy, F. D., 1998, “Staphylococcus aureusinfections.” N. Engl. J. Med, 8:520-532]. It is a common cause ofcommunity- and nosocomially-acquired septicemia (e.g., of approximately2 million infections nosocomially acquired annually in the UnitedStates, approximately 260,000 are associated with S. aureus [Emori, T.G., and R. P. Gaynes, 1993, “An overview of nosocomial infections,including the role of the microbiology laboratory,” Clin. Microbiol.Rev., 4:428-442]). Also, approximately 20% of the human population isstably colonized with S. aureus, and up to 50% of the population istransiently colonized, with diabetics, intravenous drug users, patientson dialysis, and patients with AIDS having the highest rates of S.aureus colonization [Tenover, F. C., and R. P. Gaynes, 2000, “Theepidemiology of Staphylococcus infections,” p. 414-421, In: V. A.Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, and J. I. Rood(ed), Gram-positive pathogens, American Society for Microbiology,Washington, D.C.]. The organism is responsible for approximatelyone-half of all skin and connective tissue infections, includingfolliculitis, cellulitis, furuncules, and pyomyositis, and is one of themost common causes of surgical site infections. The mortality rate forS. aureus septicemia ranges from 11 to 48% [Mortara, L. A., and A. S.Bayer, 1993, “Staphylococcus aureus bacteremia and endocarditis. Newdiagnostic and therapeutic concepts.” Infect. Dis. Clin. North. Am.,1:53-68].

Methicillin was one of the first synthetic antibiotics developed totreat penicillin-resistant staphylococcal infections. However, theprevalence of methicillin-resistant S. aureus strains or “MRSA” (whichalso are resistant to oxacillin and nafcillin) has drastically increasedin the United States and abroad [Panlilio, A. L., D. H. Culver, R. P.Gaynes, S. Banerjee, T. S. Henderson, J. S. Tolson, and W. J. Martone,1992, “Methicillin-resistant Staphylococcus aureus in U.S. hospitals,1975-1991.” Infect. Control Hosp. Epidemiol., 10:582-586]. For example,according to the National Nosocomial Infections Surveillance System[National Nosocomial Infections Surveillance (NNIS) report, data summaryfrom October 1986-April 1996, issued May 1996, “A report from theNational Nosocomial Infections Surveillance (NNIS) System.” Am. J.Infect. Control., 5:380-388], approximately 29% of 50,574 S. aureusnosocomial infections from 1987 to 1997 were resistant to the β-lactamantibiotics (e.g., oxacillin, nafcillin, methicillin), and the percentof MRSA strains among U.S. hospitals reached approximately 40% by theend of the same period. At the University of Maryland MedicalCenter, >50% of all S. aureus blood isolates are now methicillinresistant.

In this setting, there is great concern about the possible emerge ofmethicillin-resistant/multi-drug resistant S. aureus strains which arevancomycin resistant—and which would be essentially untreatable.Although overt resistance to vancomycin has not yet been documented inclinical isolates, there have been several reports of clinicalinfections with S. aureus strains having intermediate resistance tovancomycin (MICs=8 μg/ml), which suggests that untreatablestaphylococcal infections may not be too far away [Tenover, F. C., andR. P. Gaynes. 2000]. Given the virulence of S. aureus, the emergence ofsuch untreatable strains would be devastating and have a major impact onthe way in which medicine is practiced in this country.

Staphylococcal species, including MDRSA, are common colonizers of thehuman nose; in one community-based study, 35% of children and 28% oftheir guardians had nasal Staphylococcus aureus colonization (Shopsin B,et al, “Prevalence of methicillin-resistant and methicillin-susceptibleStaphylococcus aureus in the community.” J Infect Dis 2000;182:359-62.). Persons who are nasally colonized with MRSA have anincreased risk of developing serious systemic infections with thismicroorganism, and, in particular, colonization or prior infection withMDRSA significantly increases the risk of subsequent bacteremia withMDRSA (Roghmann M C, “Predicting methicillin resistance and the effectof inadequate empiric therapy on survival in patients withStaphylococcus aureus, bacteremia. Arch Intern Med 2000; 160:1001-4). Asseen with VRE, the rate of colonization of persons with MDRSA on a unit(the colonization pressure) significantly increases the risk ofacquisition of MDRSA for other patients on the unit (Merrer J, et al,““Colonization pressure” and risk of acquisition ofmethicillin-resistant Staphylococcus aureus in a medical intensive careunit.” infect Control Hosp Epidemiol 2000; 21:718-23).

Multi-Drug Resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa is a highly virulent gram-negative bacterialspecies that is responsible for bacteremia, wound infections, pneumonia,and urinary tract infections. Increasing problems with multi-antibioticresistance in Pseudomonas has been noted in hospitals, with particularconcern focusing on strains which are generally designated as“Imipenem-resistant Pseudomonas”, reflecting the last majorantimicrobial agent to which they have become resistant. Many of thesestrains are resistant to all major antibiotic classes, presentingsubstantive difficulties in management of infected patients.

As seen with other Gram-negative microorganisms, Pseudomonas strainsoften emerge as the primary colonizing flora of the posterior pharynxduring hospitalization. Strains present in the posterior pharynx, inturn, are more likely to be aspirated into the lungs, and pausepneumonia. In this setting, colonization with multi-drug resistantPseudomonas represents a potentially serious risk factor for developmentof multi-drug resistant Pseudomonas pneumonia.

Bacteriophage

Bacteriophage has been used therapeutically for much of this century.Bacteriophage, which derive their name from the Greek word “phago”meaning “to eat” or “bacteria eaters”, were independently discovered byTwort and independently by D'Herelle in the first part of the twentiethcentury. Early enthusiasm led to their use as both prophylaxis andtherapy for diseases caused by bacteria. However the results from earlystudies to evaluate bacteriophage as antimicrobial agents were variabledue to the uncontrolled study design and the inability to standardizereagents. Later in well designed and controlled studies it was concludedthat bacteriophage were not useful as antimicrobial agents (Pyle, N. J.(1936), J. Bacteriol., 12:245-61; Colvin, M. G. (1932), J. Infect Dis.,51:17-29; Boyd et al. (1944), Trans R. Soc. Trop. Med. Hyg., 37:243-62).

This initial failure of phage as antibacterial agents may have been dueto the failure to select for phage that demonstrated high in vitro lyticactivity prior to in vivo use. For example, the phage employed may havehad little or no activity against the target pathogen, were used againstbacteria that were resistant due to lysogenization or the phage itselfmight be lysogenic for the target bacterium (Barrow, et al. (1997),“Bacteriophage therapy and prophylaxis: rediscovery and renewedassessment of potential.” Trends in Microbiology, 5:268-71). However,with a better understanding of the phage-bacterium interaction and ofbacterial virulence factors, it was possible to conduct studies whichdemonstrated the in vivo anti-bacterial activity of the bacteriophage(Asheshov, et al. (1937), Lancet, 1:319-20; Ward, W. E. (1943), J.Infect. Dis., 72:172-6; Lowbury, et al. (1953), J: Gen. Microbiol.,9:524-35). In the U.S. during the 1940's Eli Lilly commerciallymanufactured six phage products for human use including preparationstargeted towards staphylococci, streptococci and other respiratorypathogens.

With the advent of antibiotics, the therapeutic use of phage graduallyfell out of favor in the U.S. and Western Europe and little subsequentresearch was conducted. However, in the 1970's and 1980's there werereports of bacteriophage therapy continuing to be utilized in EasternEurope, most notably in Poland and the former Soviet Union.

Phage therapy has been used in the former Soviet Union and EasternEurope for over half a century, with research and production centered atthe Eliava Institute of Bacteriophage in Tbilisi, in what is now theRepublic of Georgia. The international literature contains severalhundred reports on phage therapy, with the majority of the publicationscoming from researchers in the former Soviet Union and eastern Europeancountries. To give but a few examples, phages have been reported to beeffective in treating (i) skin and blood infections caused byPseudomonas, Staphylococcus, Klebsiella, Proteus, and E. coli [Cislo,M., M. Dabrowski, B. Weber-Dabrowska, and A. Woyton, 1987,“Bacteriophage treatment of suppurative skin infections,” 35(2):175-183;Slopek, S., I. Durlakowa, B. Weber-Dabrowska, A. Kucharewicz-Krukowska,M. Dabrowski, and R. Bisikiewicz, 1983, “Results of bacrteriophagetreatment of suppurative bacterial infections. I. General evaluation ofthe results,” Archivum. Immunol. Therapiae Experimental, 31:267-291;Slopek, S., B. Weber-Dabrowska, M. Dabrowski, and A.Kucharewicz-Krukowska, 1987, “Results of bacteriophage treatment ofsuppurative bacterial infections in the years 1981-1986,”, 35:569-83],(ii) staphylococcal lung and pleural infections [Meladze, G. D., M. G.Mebuke, N. S. Chkhetia, N. I. Kiknadze, G. G. Koguashvili, I. I.Timoshuk, N. G. Larionova, and G. K. Vasadze, 1982, “The efficacy ofStaphylococcal bacteriophage in treatment of purulent diseases of lungsand pleura,” Grudnaya Khirurgia, 1:53-56 (in Russian, summary inEnglish)], (iii) P. aeruginosa infections in cystic fibrosis patients[Shabalova, I. A., N. I. Karpanov, V. N. Krylov, T. O. Sharibjanova, andV. Z. Akhverdijan. “Pseudomonas aeruginosa bacteriophage in treatment ofP. aeruginosa infection in cystic fibrosis patients,” abstr. 443. InProceedings of IX international cystic fibrosis congress, Dublin,Ireland], (iv) neonatal sepsis [Pavlenishvili, I., and T. Tsertsvadze.1985. “Bacteriophage therapy and enterosorbtion in treatment of sepsisof newborns caused by gram-negative bacteria.” In abstracts, p. 104,Prenatal and Neonathal Infections, Toronto, Canada], and (v) surgicalwound infections [Peremitina, L. D., E. A. Berillo, and A. G. Khvoles,1981, “Experience in the therapeutic use of bacteriophage preparationsin supportive surgical infections.” Zh. Mikrobiol. Epidemiol.Immunobiol. 9:109-110 (in Russian)]. Several reviews of the therapeuticuse of phages were published during the 1930s-40s [Eaton, M. D., and S.Bayne-Jones, 1934, “Bacteriophage therapy: review of the principles andresults of the use of bacteriophage in the treatment of infections,” J.Am. Med. Assoc., p. 103; Krueger, A. P., and E. J. Scribner, 1941, “Thebacteriophage: its nature and its therapeutic use,” J. Am. Med. Assoc.,p. 116] and recently [Barrow, P. A., and J. S. Soothill, 1997,“Bacteriophage therapy and propylaxis—rediscovery and renewed assessmentof potential,” Trends in Microbiol., 5(7):268-271; Lederberg, J., 1996,“Smaller fleas . . . ad infinitum: therapeutic bacteriophage,” Proc.Natl. Acad. Sci. USA, 93:3167-3168]. In a recent paper published in theJournal of Infection (Alisky, J., K. Iczkowski, A. Rapoport, and N.Troitsky, 1998, “Bacteriophages show promise as antimicrobial agents,”J. Infect., 36:545), the authors reviewed Medline citations (publishedduring 1966-1996) of the therapeutic use of phages in humans. There weretwenty-seven papers from Britain, the U.S.A., Poland and the SovietUnion, and they found that the overall reported success rate for phagetherapy was in the range of 80-95%.

These are several British studies describing controlled trials ofbacteriophage raised against specific pathogens in experimentallyinfected animal models such as mice and guinea pigs (See, e.g., Smith.H. W., and M. B. Huggins “Successful treatment of experimentalEscherichia coli infections in mice using phages: its generalsuperiority over antibiotics” J. Gen. Microbial., 128:307-318 (1982);Smith, H. W., and M. B. Huggins “Effectiveness of phages in treatingexperimental E. coli diarrhea in calves, piglets and lambs” J. Gen.Microbial., 129:2659-2675 (1983); Smith, H. W. and R. B. Huggins “Thecontrol of experimental E. coli diarrhea in calves by means ofbacteriophage”. J. Gen. Microbial., 133:1111-1126 (1987); Smith, H. W.,R. B. Huggins and K. M. Shaw “Factors influencing the survival andmultiplication of bacteriophages in calves and in their environment” J.Gen. Microbial., 133:1127-1135 (1987)). These trials measured objectivecriteria such as survival rates. Efficacy against Staphylococcus,Pseudomonas and Acinetobacter infections were observed. These studiesare described in more detail below.

One U.S. study concentrated on improving bioavailability of phage inlive animals (Merril, C. R., B. Biswas, R. Carlton, N. C. Jensen, G. J.Greed, S. Zullo, S. Adhya “Long-circulating bacteriophage asantibacterial agents” Proc. Natl. Acad Sci. USA, 93:3188-3192 (1996)).Reports from the U.S. relating to bacteriophage administration fordiagnostic purposes have indicated phage have been safely administeredto humans in order to monitor humoral immune response in adenosinedeaminase deficient patients (Ochs, et al. (1992), “Antibody responsesto bacteriophage phi X174 in patients with adenosine deaminasedeficiency.” Blood, 80:1163-71) and for analyzing the importance of cellassociated molecules in modulating the immune response in humans (Ochs,et al. (1993), “Regulation of antibody responses: the role of complementacrd adhesion molecules.” Clin. Immunol. Immunopathol., 67:S33-40).

Additionally, Polish, Georgian, and Russian papers describe experimentswhere phage was administered systemically, topically or orally to treata wide variety of antimicrobial resistant pathogens (See, e.g.,Shabalova, I. A., N. I. Karpanov, V. N. Krylov, T. O. Sharibjanova, andV. Z. Akhverdijan. “Pseudomonas aeruginosa bacteriophage in treatment ofP. aeruginosa infection in cystic fibrosis patients,” Abstr. 443. InProceedings of IX International Cystic Fibrosis Congress, Dublin,Ireland; Slopek, S., I. Durlakowa, B. Weber-Dabrowska, A.Kucharewicz-Krukowska, M. Dabrowski, and R Bisikiewicz. 1983. “Resultsof bacteriophage treatment of suppurative bacterial infections. I.General evaluation of the results.” Archivum Immunol. TherapiaeExperimental, 31:267-291; Slopek, S., B. Weber-Dabrowska, M. Dabrowski,and A. Kucharewicz-Krukowska. 1987. “Results of bacteriophage treatmentof suppurative bacterial infections in the years 1981-1986”, ArchivumImmunol. Therapiae Experimental, 35:569-83.

Infections treated with bacteriophage included osteomyelitis, sepsis,empyema, gastroenteritis, suppurative wound infection, pneumonia anddermatitis. Pathogens involved included Staphylococci, Sreptococci,Klebsiella, Shigella, Salmonella, Pseudomonas, Proteus and Escherichia.These articles reported a range of success rates for phage therapybetween 8095% with only rare reversible allergic or gastrointestinalside effects. These results indicate that bacteriophage may be a usefuladjunct in the fight against bacterial diseases. However, thisliterature does not describe, in any way anticipate, or otherwisesuggest the use of bacteriophage to modify the composition of colonizingbacterial flora in humans, thereby reducing the risk of subsequentdevelopment of active infections.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, a lyticbacteriophage which infects Enterococcus, wherein no more than 30% ofthe Enterococcus strains in a collection of more than 100 geneticallydiverse vancomycin resistant Enterococcus (VRE) strains are resistant toinfection by said bacteriophage is disclosed. The bacteriophage mayproduces lytic infection in at least 200 genetically diverse VREisolates. The bacteriophage preparation may be administered orally topatients who are colonized with VRE who are admitted to a medicalfacility. These patients may include, inter alia, leukemia patients,lymphoma patient, transplant patients and AIDS patients. Thebacteriophage may also be administered to all patients admitted at themedical facility. Administration of this bacteriophage will reduce oreliminate colonization with VRE, thereby reducing the risk that thesepatients will subsequently develop serious systemic infections with thishighly resistant microorganism. Reduction or elimination of colonizationwill also decrease the VRE “colonization pressure” in the hospital orspecific units of the hospital, thereby reducing the risk that VRE willbe acquired by persons who currently are neither colonized or infectedwith the pathogen.

In one embodiment, the present invention provides a method for reducingthe risk of bacterial infection or sepsis in a susceptible patient bytreating the susceptible patient with a pharmaceutical compositioncontaining bacteriophage of one or more strains which produce lyticinfections in pathogenic bacteria. Preferably, treatment of the patientreduces the level of colonization with pathogenic bacteria susceptibleto the bacteriophage by at least one log. In a typical embodiment, thesusceptible patient is an immunocompromised patient selected from thegroup consisting of leukemia patients, lymphoma patients, carcinomapatients, sarcoma patients, allogeneic transplant patients, congenitalor acquired immunodeficiency patients, cystic fibrosis patients, andAIDS patients. In a preferred mode, the patients treated by this methodare colonized with the pathogenic bacteria subject to infection by saidbacteriophage.

In a preferred embodiment of this invention, the risk of infection isreduced by administering a composition comprising bacteriophage whichproduce lytic infections in pathogenic bacteria selected fromvancomycin-resistant enterococcus (VRE), pneumococcal species,methicillin-resistant Staphylococcus aureus, multi-drug resistantStaphylococcus aureus (MDRSA), multi-drug resistant Pseudomonas species,Nesseria sp., Hemophilus sp., Proteus sp., Klebsiella sp. and Esherichiacoli. Preferably, the pathogenic bacteria are selected from VRE, MDSA,and multi-drug resistant Pseudomonas. In a preferred embodiment of thisinvention, the bacteriophage composition is in a form selected from aparenteral composition, an oral tablet, capsule or liquid, a nasalaerosol, a throat wash, a toothpaste, and a topical ointment.Preferably, the pharmaceutical composition contains a plurality ofbacteriophage strains. More preferably, the pharmaceutical compositioncontains bacteriophage strains which produce lytic infections inpathogenic bacteria of a plurality of bacterial strains or bacteriophagestrains which produce lytic infections in pathogenic bacteria of aplurality of bacterial species.

In a preferred embodiment, the present invention provides a method forreducing the risk of bacterial infection or sepsis in a patient having awound selected from an ulcer, a laceration, a deep penetrating wound anda surgical wound by treating the patient with a pharmaceuticalcomposition containing bacteriophage of one or more strains whichproduce lytic infections in pathogenic bacteria capable of infectingthese wounds. Preferably, the composition is a topical ointment, anirrigation solution or a component of a wound dressing.

In another embodiment, this invention provides a method for reducing theincidence of infection by selected bacteria in a medical facility byadministering to patients who are admitted to said medical facility abacteriophage preparation which reduces the colonization level by theselected bacteria in patients at risk for infection by the selectedbacteria. In a typical embodiment, the patients at risk for infectionare selected from the group consisting of leukemia patients, lymphomapatients, carcinoma patients, sarcoma patients, allogeneic transplantpatients, congenital or acquired immunodeficiency patients, cysticfibrosis patients, and AIDS patients. In another embodiment, thebacteriophage preparation is administered to substantially all patientsadmitted to said medical facility. In a preferred embodiment, thebacteriophage preparation is administered to substantially all patientscolonized with the selected bacteria who are admitted to said medicalfacility. In another preferred embodiment, the selected bacteria is VRE,MDRSA, or multi-drug resistant Pseudomonas.

According to another embodiment of the present invention, abacteriophage preparation which reduces the number of VRE inexperimentally infected mice by at least 1 log is disclosed.

According to another embodiment of the present invention, a lyticbacteriophage which infects Staphylococcus aureus, wherein no more than30% of the Staphylococcal strains in a collection of more than 100genetically diverse multi-drug resistant Staphylococcus aureus (MDRSA)strains are resistant to infection by said bacteriophage is disclosed.The bacteriophage may produces lytic infection in at least 200genetically diverse MDRSA isolates. The bacteriophage preparation may beadministered via nasal spray to individuals who are nasally colonizedwith MDRSA, particularly to a subpopulation made up of all suchindividuals who are admitted to a medical facility. The bacteriophagemay also be administered to all patients admitted at the medicalfacility. Administration of this bacteriophage will reduce or eliminatecolonization with MDRSA, thereby reducing the risk that these patientswill subsequently develop serious systemic infections with this highlyresistant microorganism. Reduction or elimination of colonization willalso decrease the MDRSA “colonization pressure” in the hospital orspecific units of the hospital, thereby reducing the risk that MDRSAwill be acquired by persons who currently are neither colonized orinfected with the pathogen.

According to one embodiment of the present invention, a lyticbacteriophage which infects Pseudomonas aeruginosa, wherein no more than30% of the Pseudomonas strains in a collection of more than 100genetically diverse multi-antibiotic resistant Pseudomonas aeruginosastrains are resistant to infection by said bacteriophage is disclosed.The bacteriophage may produces lytic infection in at least 200genetically diverse isolates. The bacteriophage preparation may beadministered by mouth wash or gargle to individuals who are colonizedwith multi-drug resistant Pseudomonas aeruginosa, particularly to asubpopulation made up of all such individuals who are admitted to amedical facility. The bacteriophage may also be administered to allpatients admitted at the medical facility. Administration of thisbacteriophage will reduce or eliminate colonization with multi-drugresistant Pseudomonas strains, thereby reducing the risk that thesepatients will subsequently develop serious systemic infections with thishighly resistant microorganism. This may also be expected to reduce the“colonization pressure” of this microorganism, thereby reducing the riskthat it will be acquired by persons who are currently not colonized orinfected.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Bacteriophage technology can be of value in managing a large variety ofbacterial infections because: (i) bacteriophages are highly specific andvery effective in lysing targeted pathogenic bacteria, (ii)bacteriophages arc absolutely specific for prokaryotes, and do notaffect humans or animals, (iii) bacteriophages are safe, as underscoredby their extensive clinical use in Eastern Europe and the former SovietUnion, and the commercial sale of phages in the 1940's in the UnitedStates, (iv) phage preparations can rapidly be modified to combat theemergence of newly arising bacterial threats, and (v) phage productionis seen to be cost-effective for large-scale applications in a varietyof medical settings. Of particular relevance, bacteriophage will notkill non-pathogenic, “normal flora” bacteria, thereby retaining the“colonization resistance” of reservoirs such as the human intestinaltract, the nose, and the posterior pharynx. Accordingly, the presentinvention envisions using lytic phages (in combination with antibioticsor alone) to prophylactically or therapeutically eliminate variousbacteria capable of causing diseases of the gastrointestinal,genitourinary, and respiratory tracts, and skin, oral cavity, andbloodstream. In accordance with this invention, therapeutic phages canbe administered in a number of ways, in various formulations, including:(i) orally, in tablets or liquids, (ii) locally, in tampons, rinses orcreams, (iii) aerosols, and (iv) intravenously.

One benefit of bacteriophage therapy when compared to antibiotic therapyrelates to the relative specificity of the two therapeutic modalities.Bacteriophage are specific for particular bacterial strains or species,while antibiotics typically arc broadly effective against a largemultiplicity of bacterial species or genera. It is well known thatnormal individuals are colonized with innocuous bacteria, and thiscolonization may be beneficial to the colonized individual (see U.S.Pat. No. 6,132,710, incorporated herein by reference). Antibiotictherapy can severely alter colonization or even eliminate beneficialcolonization completely. This may have adverse effects, such as theoutgrowth of opportunistic species such as Clostridium difficile, whichthen leads to an antibiotic associated colitis. In contrast,bacteriophage therapy specifically affects the bacterial strains thatare sensitive or susceptible to lytic infection by the particularbacteriophage in the therapeutic composition, but leaves other(innocuous or beneficial) bacteria unaffected. Thus, bacteriophagetherapy is preferable for prophylactic treatment where alteration ofnormal microflora should be minimized.

In a preferred mode of this invention, phage technology is focused ontwo important human pathogens, VRE and MDRSA, and the value of VRE- andMDRSA-specific lytic phages in different settings: (i) oraladministration of phages for prophylaxis against septicemia, (ii) localapplication of phages for prophylaxis/treatment of skin and woundinfections, (iii) intravenous administration of phages for therapy ofsepticemia, and (iv) the use of aerosolized phages against respiratorypathogens.

VRE infection has become a particularly serious problem amongimmunocompromised and/or seriously ill patients in intensive care units,cancer centers and organ transplant units. Since VRE are resistant toall currently used antimicrobials, alternate approaches to reducing oreliminating VRE gastrointestinal colonization in immunocompromisedpatients must be found in order to reduce the prevalence of VREbacteremia. Oral administration of lytic bacteriophage active againstVRE is one such approach.

The general rule is that patients first become colonized by pathogenicbacteria present in their immediate environment before developingillness due to those bacteria. Serious VRE infections, includingsepticemia, usually are preceded by intestinal colonization with theinfecting organisms; therefore, the risk of septicemia is likely to bedecreased by reducing colonization prior to periods when patients areseverely neutropenic or otherwise immunosuppressed (i.e., reducingintestinal colonization may also reduce the risk of bloodstreaminvasion). The present inventors have discovered that certain strains ofbacteriophage arc particularly effective at lysing VRE. By administeringthese VRE-active bacteriophage to persons colonized with VRE, it ispossible to substantially reduce or even eliminate VRE from thecolonized person. Thus, the present invention provides strains of phagewhich are particularly effective against VRE, methods for obtainingadditional strains of VRE-active phage, methods for treating patientscolonized with VRE by administering VRE-active phage, and methods ofreducing nosicomial infection rate by administering VRE-active phage invivo, ex vivo, or both, to selected locations, areas, objects and/orpersons.

Analogous approaches using bacteriophage targeted to other pathogenicbacteria are also contemplated by this invention. S. aureus phagepreparations can reduce contamination of skin and wounds with S. aureus,which in turn may prevent the development of serious surgical siteinfections and septicemia. Phage active against Pseudomonas species canbe used to reduce colonization that threatens to develop into pneumoniain immunocompromised patients or in individuals suffering from cysticfibrosis.

Isolation of Bacteriophage

The present inventors have isolated several lytic phages active againstgenetically diverse (as assessed by pulsed field gel electrophoresisand/or arbitrary pruned polymerase chain reaction or other nucleic acidamplification techniques) VRE strains. In vitro susceptibility testsinvolving 234 VRE strains (184 E. faecium, 41 E. faecalis and 6 E.gallinarium isolated from patients at the University of Maryland and theBaltimore VA Medical Center, and 3 E. faecium ATCC strains), resulted inthe Intralytix phage collection being able, to cumulatively lyse all VREstrains in the collection, with one particular phage being able to lyse95% of VRE strains. Furthermore mice whose gastrointestinal tract wascolonized with VRE under selective pressure of antibioticadministration, were orogastrically administered VRE-active phages,which resulted in a 1 to 3 log reduction of VRE gastrointestinalcolonization compared to a control group of animals not given phage.This occurred within a 48 to 72 hour time frame. No side effects due tothe phage were observed.

Bacteriophage strains may be isolated by analogous procedures to thoseused to isolate the VRE-active strains described herein. Suitablebacteriophage may be isolated from any sample containing bacteriophage,which typically are found in association with their host bacteria. Thus,any source that might be expected to contain VRE is suitable for use asa source of VRE-active bacteriophage. Such samples include fecal, urine,or sputum samples from patients, particularly patients undergoing acuteor prophylactic antibiotic therapy, patients in intensive care units orimmunocompromised patients. Such patients may include but are notlimited to burn patients, trauma patients, patients receiving bonemarrow and/or organ transplants, cancer patients, patients withcongenital or acquired immunodeficiency diseases, dialysis patients,liver disease patients, and patients with acute or chronic renalfailure. Body fluids including ascites, pleural effusions, jointeffusions, abscess fluids, and material obtained from wounds. Whilehumans are the primary reservoir for VRE, the organism also can bereadily found in the immediate environment of infected/colonizedpatients such as bedrails, bed sheets, furniture, etc. (Bodnar, U. R. etal (1996), “Use of in house studies of molecular epidemiology and fullspecies identification of controlling spread of vancomycin resistantEnterococcus faecalis isolates”, J. Clin. Microbial., 34: 2129-32;Bonten, M. J. M. et al (1996), “Epidemiology of colonization of patientsand the environment with vancomycin resistant enterococci.” Lancet, 348:1615-19; Noskin, G. A. (1995), “Recovery of vancomycin resistantenterococci on fingertips and environmental surfaces.” Infect. ControlHosp. Epidemiol., 16: 577-81). Consequently, samples for bacteriophageisolation may also be obtained from nonpatient sources, includingsewage, especially sewage streams near intensive care units or otherhospital venues, or by swab in hospital areas associated with risk ofnosicomial infection, such as intensive care units. Other suitablesampling sites include nursing homes, rest homes, military barracks,dormitories, classrooms, and medical waste facilities. Phages also canbe isolated from rivers and lakes, wells, water tables, as well as otherwater sources (including salt water). Preferred sampling sites includewater sources near likely sites of contamination listed above.

Suitable methods for isolating pure bacteriophage strains from abacteriophage-containing sample are well known, and such methods may beadapted by the skilled artisan in view of the guidance provided herein.Isolation of VRE-active bacteriophage from suitable samples typicallyproceeds by mixing the sample with nutrient broth, inoculating the brothwith a host bacterial strain, and incubating to enrich the mixture withbacteriophage that can infect the host strain. An Enterococcus sp.strain will be used as the host strain, preferably a VRE strain. Afterthe incubation for enrichment, the mixture is filtered to removebacterial leaving lytic bacteriophage in the filtrate. Serial dilutionsof the filtrate are plated on a lawn of VRE, and VRE-active phage infectand lyse neighboring bacteria. However the agar limits the physicalspread of the phage throughout the plate, resulting in small visiblyclear areas called plaques on the plate where bacteriophage hasdestroyed VRE within the confluent lawn of VRE growth. Since one plaquewith a distinct morphology represents one phage particle that replicatedin VRE within that area of the bacterial lawn, the purity of abacteriophage preparation can be ensured by removing the material inthat plaque with a pasteur pipette (a “plaque pick”) and using thismaterial as the inoculum for further growth cycles of the phage. Thebacteriophage produced in such cycles represent a single strain or“monophage.” The purity of phage preparation (including confirmationthat it is a monophage and not a polyvalent phage preparation) isassessed by a combination of electron microscopy, SDS-PAGE, DNArestriction digest and analytical ultracentrifugation. In addition, eachphage is uniquely identified by its DNA restriction digest profile,protein composition, and/or genome sequence.

Individual VRE-active bacteriophage strains (i.e., monophages) arepropagated as described for enrichment culture above, and then testedfor activity against multiple VRE strains to select broad-spectrumVRE-active bacteriophage. Efforts are made to select phages that (i) arelytic, (ii) are specific to enterococci, (iii) lyse more than 70% of theVRE strains in our VRE strain collection, and/or (iv) lyse VRE strainsresistant to other VRE phages previously identified. It is also possibleto select appropriate phages based upon the sequences of DNA or RNAencoding proteins involved in the binding and/or entry of phage intotheir specific host, or based upon the amino acid sequences or antigenicproperties of such proteins.

Quantities of broad-spectrum VRE-active bacteriophage needed fortherapeutic uses described below may be produced by culture on asuitable host strain in the mariner described above for enrichmentculture. When performing an enrichment culture to produce bacteriophagefor therapeutic use, a host strain is selected based on its ability togive a maximum yield of phage, as determined in pilot experiments withseveral different host VRE strains. If two or more host strains givesimilar yield' the strain most sensitive to antibiotics is selected.

The techniques described herein for isolation of VRE monophages areapplicable to isolation of bacteriophages that are lytic for otherpathogenic bacteria. It is within the skill in the art to substitutehost strains of other bacteria in the methods described herein in orderto isolate phage specific for those bacteria.

Starting the phage isolation process with samples selected fromenvironments that also contain bacteria of the host species willaccelerate the process.

Patient Population

Any patient who is at risk for colonization with VRE or who has provenVRE colonization is a candidate for treatment according to the method ofthis invention. Intestinal colonization with VRE is relatively common ininstitutionalized patients undergoing antimicrobial therapy. In studiesconducted in 1993-94, 17-19% of a random sample of all patients at theUniversity of Maryland Hospital were colonized with VRE (Morris, et al.(1995), “Enterococci resistant to multiple antimicrobial agentsincluding vancomycin.” Ann. Int. Med., 123:250-9), while in an identicalstudy conducted in 1996 this increased to 23.8%. Once colonized withVRE, a patient may remain colonized for life; however once offantimicrobial therapy, VRE colonization may drop to levels notdetectable in routine stool culture. Colonized persons though who alsosubsequently become immunocompromised are at risk for developingbacteremia (Edmond, et al., 1995; Tornieporth, et al (1996), “Riskfactors associated with vancomycin resistant Enterococcus faeciumcolonization or infection in 145 matched case patients and controlpatients.” Clin. Infect. Dis., 23:767-72).

VRE infection is a particularly serious problem among immunocompromisedand/or seriously ill patients in cancer centers, intensive care units,and organ transplant centers. In case control studies VRE has beenlinked to antimicrobial use and severity of illness (as measured byAPACHE score) (Handwerger, et al. (1993), “Nosocomial outbreak due toEnterococcus faecium, highly resistant to vancomycin, penicillin andgentamicin.” Clin. Infect. Dis., 16:750-5; Montecalvo, et al. (1996),“Bloodstream infections with vancomycin resistant enterococci.” Arch.Intern. Med., 156:1458-62; Papanicolaou, et al. (1996), “Nosocomialinfections with vancomycin-resistant Enterococcus faecium in livertransplant patients: Risk factors for acquisition and mortality.” Clin.Infect. Dis., 23:760-6; Roghmann, et al., (1997), “Recurrent vancomycinresistant Enterococcus faecium bacteremia in a leukemic patient who waspersistently colonized with vancomycin resistant enterococci for twoyears.” Clin. Infect. Dis., 24:514-5). Investigators at the Universityof Maryland at Baltimore and the Baltimore VA Medical Center havedemonstrated by pulse field electrophoresis that VRE strains causingbacteremia in cancer patients are almost always identical to those thatcolonize the patient's gastrointestinal tract.

Three categories of immunocompromised patients subjected to prolongedantimicrobial administration in a institutionalized setting and whowould be susceptible to VRE gastrointestinal colonization are: 1)leukemia (30,200 patients per year in the U.S.) and lymphoma patients(64,000 patients per year in the U.S.), 2) transplant patients (20,961per year in the U.S.), and 3) AIDS patients (66,659 patients per year inthe U.S.). The total number of patients in the immunocompromisedcategory is 181,800 per year in the U.S. Pfundstein, et al., found thatthe typical rate of enterococcal gastrointestinal colonization amongrenal and pancreas transplant patients receiving antibiotics in aninstitutional setting was 34% (38/102) with 4 (11%) of these isolatesbeing VRE (Pfundstein, et al. (1999), “A randomized trial of surgicalantimicrobial prophylaxis with and without vancomycin in organtransplant patients.” Clin. Transplant., 13:245-52). Therefore the rateof gastrointestinal colonization by VRE in this immunocompromisedpopulation would be 0.34×0.11=0.04 or 4% of the total patientpopulation. One can therefore estimate VRE gastrointestinal,colonization to be 181,800×0.04=7272 patients per year.

Formulation and Therapy

According to this invention, VRE-active bacteriophage are preferablyformulated in pharmaceutical compositions containing the bacteriophageand a pharmaceutically acceptable carrier, and can be stored as aconcentrated aqueous solution or lyophilized powder preparation.Bacteriophage may be formulated for oral administration by resuspendingpurified phage preparation in aqueous medium, such as deionized water,mineral water, 5% sucrose solution, glycerol, dextran, polyethyleneglycol, sorbitol, or such other formulations that maintain phageviability, and are non-toxic to humans. The pharmaceutical compositionmay contain other components so long as the other components do notreduce the effectiveness (ineffectivity) of the bacteriophage so muchthat the therapy is negated. Pharmaceutically acceptable carriers arewell known, and one skilled in the pharmaceutical art can easily selectcarriers suitable for particular routes of administration (Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).

The pharmaceutical compositions containing VRE-active bacteriophage maybe administered by parenteral (subcutaneously, intramuscularly,intravenously, intraperitoneally, intrapleurally, intravesicularly orintrathecally), topical, oral, rectal, inhalation, ocular, otic, ornasal route, as necessitated by choice of drug and disease.

Injection of specific lytic phages directly into the bloodstream caneliminate or significantly reduce the number of targeted bacteria in theblood. If, after either oral or local administration, phages get intothe bloodstream in sufficient numbers to eliminate bacteria from thebloodstream, septicemia may be treated by administering phages orally(or locally). If the phages do not get into the bloodstream insufficient numbers to eliminate bacteria from the bloodstream, theutility of direct iv. injection of phages for treating septic infectionscan be used to treat bloodstream infections caused by VRE and otherpathogenic bacteria, and can provide an urgently needed means fordealing with currently untreatable septicemic infections.

Dose and duration of therapy will depend on a variety of factors,including the patient age, patient weight, and tolerance of the page.Bacteriophage may be administered to patients in need of the therapyprovided by this invention by oral administration. Based on previoushuman experience in Europe, a dose of phage between 10⁷ and 10¹¹ PFUwill be suitable in most instances. The phage may be administered orallyin, for example, mineral water, optionally with 2.0 grams of sodiumbicarbonate added to reduce stomach acidity. Alternatively, sodiumbicarbonate may be administered separately to the patient just prior todosing with the phage. Phages also may be incorporated in a tablet orcapsule which will enable transfer of phages through the stomach with noreduction of phage viability due to gastric acidity, and release offully active phages in the small intestine. The frequency of dosing willvary depending on how well the phage is tolerated by the patient and howeffective a single versus multiple dose is at reducing VREgastrointestinal colonization.

The dose of VRE-active bacteriophage and duration of therapy for aparticular patient can be determined by the skilled clinician usingstandard pharmacological approaches in view of the above factors. Theresponse to treatment may be monitored by, analysis of blood or bodyfluid levels of VRE, or VRE levels in relevant tissues or monitoringdisease state in the patient. The skilled clinician will adjust the doseand duration of therapy based ors the response to treatment revealed bythese measurements.

One of the major concerns about the use of phages in clinical settingsis the possible development of bacterial resistance against them.However, as with antimicrobial resistance, the development of resistanceto phages takes time. The successful use of phages in clinical settingswill require continual monitoring for the development of resistance,and, when resistance appears, the substitution of other phages to whichthe bacterial mutants are not resistant. In general, phage preparationsmay be constructed by mixing several separately grown andwell-characterized lytic monophages, in order to (i) achieve thedesired, broad target activity of the phage preparation, (ii) ensurethat the preparation has stable lytic properties, and (iii) minimize thedevelopment of resistance against the preparation.

The development of neutralizing antibodies against a specific phage alsois possible, especially after parenteral administration (it is less of aconcern when phages are administered orally and/or locally). However,the development. of neutralizing antibodies may not pose a significantobstacle in the proposed clinical settings, because the kinetics ofphage action is much faster than is the host production of neutralizingantibodies. For VRE for example, phages will be used for just a fewdays, sufficient to reduce VRE colonization during the time period whenimmunocompromised patients are most susceptible to the development ofpotentially fatal VRE septicemia, but not long enough forphage-neutralizing antibodies to develop. If the development ofantiphage antibodies is a problem, several strategies can be used toaddress this issue. For example, different phages having the samespectrum of activity (but a different antigenic profile) may beadministered at different times during the course of therapy. On a moresophisticated level, therapeutic phages may, be genetically engineeredwhich will have a broad lytic range and/or be less immunogenic in humansand animals.

Environmental Therapy

In the 1980's a number of British studies were conducted whichdemonstrated the efficacy of bacteriophage prophylaxis and therapy inmice and farm animal models. These studies were significant because thetiters of the phage preparations administered were significantly lessthan the bacterial inoculum indicating in vivo bacteriophagemultiplication. For example, Smith et al (Smith, et al. (1982),“Successful treatment of experimental Escherichia coli infections inmice using phage: its general superiority over antibiotics.” J. Gen.Microbiol., 128:307-1825) found intra-muscular inoculation of mice with10⁶ CFU of E. coli with K1 capsule killed 10/10 mice. However when micewere simultaneously intramuscularly inoculated with 10⁴ PFU of phage, ata separate site, 10/10 mice survived. Smith and coworkers demonstratedthat administration of a mixture of two phage resulted in high levels ofprotection of calves with diarrhea induced by E. coli with K 88 or K99fimbriae (Smith, et al. (1983), “Effectiveness of phages in treatingexperimental Escherichia coli diarrhea in calves, piglets and lambs.” J.Gen. Microbial., 129:2659-75; Smith, et al. (1987), “The control ofexperimental Escherichia coli diarrhea in calves by means ofbacteriophage.” J. Gen. Microbiol., 133:1111-26; Smith, et al. (1987),“Factors influencing the survival and multiplication of bacteriophagesin calves and in their environment.” J. Gen. Microbial., 133:1127-35).If the phage was administered before or at tire same time as E. coli nodeaths occurred and complete protection was attained. Control animalsdeveloped watery diarrhea and died within 2 to 5 days. If phageadministration was delayed until the onset of diarrhea, protection wasnot complete although the severity of infection was greatly reduced andno deaths were observed. Berchieri, et al., found that fewer chicksorally infected with 10⁹ PFU of Salmonella typhimurium died when 10⁹ PFUof Salmonella specific phage was orally administered soon afterinitiation of the bacterial infection (Berchieri, et al. (1991), “Theactivity in the chicken alimentary tract of bacteriophages lytic forSalmonella typhimurium.” Res. Microbial., 142:541-49). They also foundthat the phage was readily spread between the different infected birds.

Environmental applications of phage in health care institutions couldlie most useful for equipment such as endoscopes and environments suchas ICUs which maybe potential sources of nosocomial infection due topathogens such as VRE but which may be difficult or impossible todisinfect. Phage would be particularly useful in treating equipment orenvironments inhabited by bacterial genera such as Pseudomonas which maybecome resistant to commonly used disinfectants. In the Soviet Unionthere has been a report that application of phage to the hospitalenvironment has resulted in killing targeted bacteria such asStaphylococci and Pseudomonas within 48-72 hours. Phage persisted in theenvironment as long as there were target bacteria present and uponelimination of target bacteria, phage became undetectable in 6-8 days(Alavidze, et al, 1988, “Use of specific bacteriophage in theprophylaxis of intrahospital infections caused by P. aeruginosa.” inAbstracts, All-Soviet Union conference “Modern biology at the service ofpublic health”, Kiev, Ukraine).

Phase compositions used to disinfect inanimate objects or theenvironment may be sprayed, painted, or poured, onto such objects orsurfaces in aqueous solutions with phage titers ranging between 10⁷-10¹¹PFU/ml. Alternatively, phage may be applied by aerosolizing agents thatmight include dry dispersants which would facilitate distribution of thephage into the environment. Such agents may also be included in thespray if compatible with phage viability and nontoxic in nature.Finally, objects may be immersed in a solution containing phage. Theoptimal numbers and timing of applications of phage compositions remainsto be determined and would be predicated by the exact usage of suchproducts.

Since phage are normally widely present in the environment and are foundeven in food or drugs, there is minimal safety concern with regard toapplying phage preparations to the environment.

As reported above, Smith and Huggins in England found that E. coliinduced diarrhea in calves could be prevented by simply spraying thelitter in the calf rooms with an aqueous phage preparation or even bykeeping the calves in uncleaned rooms previously occupied by calveswhose E. coli infections had been treated with phage. There is also datafrom the Soviet Union indicating the efficacy of phage to rid chickenhouses of Staphylococci (Ponomarchuk, et al., (1987), “Strain phageStaphylococci applicable for prophylaxis and therapy of poultryStaphylococcus.” Soviet patent N1389287, Dec. 15, 1987).

In the future, application of VRE phage to the environment of farmanimals such as chickens or cattle maybe necessary to reduce VRE in thissetting if VRE become prevalent in such environments and such animal VREare capable, upon being consumed in contaminated food, of transientlycolonizing the human gastrointestinal tract long enough to transferantibiotic resistance gene transposons to normal gut flora (Latta, S.(1999) “Debate heats up over antibiotic-resistant foodborne bacteria.”The Scientist 13; (14) 4-5).

Alternatively, colonization in the farm animals may be reduced byadministering bacteriophage to the animals (or treating the animals'environment) using phage that produce lytic infections in targetbacteria which colonize the animals. Such unwanted colonization may be aparticular problem with intensive agricultural techniques, such as thoseused for chickens or veal calves. Target organisms include Salmonellasp. and E. coli H7:O157.

Bacteriophage Cocktails

This invention also contemplates phage cocktails which may be customtailored to the pathogens that are prevalent in a certain situation.Typically, pathogenic bacteria would be initially isolated from aparticular source (e.g., a patient or location contaminated with VRE)and susceptibility testing of the pathogens to various bacteriophagestrains would be performed, analogous to antimicrobial susceptibilitytesting. Once each pathogen's phage susceptibility profile isdetermined, the appropriate phage cocktail can be formulated from phagestrains to which the pathogens are susceptible and administered to thepatient. Since phage would often be used in institutional settings wherepathogens are resistant to many antimicrobial agents, phage cocktailswould often consist of phage lytic for the most prevalent institutionalpathogens which, in addition to enterococci, are Staphylococcus aureus,Staphylococcus epidermidis, E. coli and Pseudomonas aeruginosa. Alsosince enterococci are often involved in polymicrobial infections alongwith other gastrointestinal commensals, such as in pelvic woundinfections, the approach of therapeutically using cocktails of phagelytic against different bacterial species would be most appropriate.Since phage cocktails would be constructed of phage againstinstitutional pathogens, isolation of such phage would be mostsuccessful from the sewage of such institutions. Typically, the phagecocktail will include one or more VRE-active bacteriophage according tothis invention.

It may be appropriate to use certain phage cocktails in agriculturalsettings where there are certain human pathogens such as Salmonella andCampylobacter inherent to poultry or livestock and which contaminate theenvironment of such animals on an ongoing basis. The result is acontinuing source of infection by such pathogens.

Bacteriophage cocktails may be applied contemporaneously—that is, theymay be applied at the same time (e.g., in the same application), or maybe applied in separate applications spaced in time such that they areeffective at the same time. The bacteriophage may be applied as a singleapplication, periodic applications, or as a continuous application.

Other bacteria within the contemplation of the present inventioninclude, inter alia, Campylobacter, E. coli H7:0157, and Listeria, andStapholoccocus.

EXAMPLES Example 1 Obtaining VRE Isolates

Isolation of VRE

VRE were isolated by standard methods from patients in the surgicalintensive care and intermediate care units of the University of MarylandMedical Center in Baltimore. Trypticase Soy Agar supplemented with 5%sheep blood (BBL, Cockeysville Md.) was used to isolate enterococci fromurine, wounds and sterile body fluids. VRE were isolated from stoolspecimens on Colistin Nalidixic Acid (CNA) agar (Difco labs, Detroit,Mich.) supplemented with defibrinated sheep blood (5%), vancomycin (10g/ml) and amphotericin (1 g/ml). See Facklam, R. R., and D. F. Sahm.1995. Enterococcus. In: Manual of Clinical Microbiology, 6^(th) edition,American Society for Microbiology, Washington, D.C., pp. 508-312.

Identification of VRE

Enterococci were identified by esculin hydrolysis and growth in 6.5%NaCl at 45° C. Identification to the species level was done usingconventional testing as indicated in Packlam and Collins (Facklam, etal. (1989), “Identification of Enterococcus species isolated from humaninfections by a conventional method test scheme.” J. Clin. Microbiol.27:731-4).

Antimicrobial Susceptibility Testing of VRE

Antimicrobial susceptibilities to ampicillin, vancomycin, streptomycin,and gentamicin were determined using the E test quantitative minimuminhibitory concentration procedure (AB Biodisk, Solna Sweden). Qualitycontrol stains of E. faecium (ATCC 29212, 51299) were used to ensurepotency of each antimicrobial agent tested. With exception ofvancomycin, susceptibility interpretations from the National Committeefor Clinical Laboratory Standards were adhered to (National Committeefor Clinical Laboratory Procedures (1993), “Methods for DilutionAntimicrobial Susceptibility Tests for Bacteria that Grow Aerobically.”3rd Edition. National Committee for Clinical Laboratory StandardsVillanova Pa.; National Committee for Clinical Laboratory Standards(1993), “Performance Standards for Antimicrobial Disk SusceptibilityTests” 5th Edition, National Committee for Clinical LaboratoryStandards, Villanova Pa.). A VRE isolate was defined as one which had aminimum inhibitory concentration to vancomycin of at least 16 g/ml.

Defining Genetically Distinct VRE Strains

Distinct VRE isolates were characterized as such by contour-clampedhomogeneous electric field electrophoresis after digestion ofchromosomal DNA with Sinai (Verma, P. et al. (1994) “Epidemiologiccharacterization of vancomycin resistant enterococci recovered from aUniversity Hospital” (Abstract). In; Abstracts of the 94th GeneralMeeting of the American Society for Microbiology, Las Vegas Nev.; Dean,et al. (1994) “Vancomycin resistant enterococci (VRE) of the vanBgenotype demonstrating glycoprotein (G) resistance inducible byvancomycin (V) or teicoplanin (T)” In; Abstracts of the 94th GeneralMeeting of the American Society for Microbiology, Las Vegas Nev.).Electrophoretic studies were also performed using ApaI digestion for VREstrains which differed only by 1-3 bands after initial analysis(Donabedian, S. M. et al (1992) “Molecular typing ofampicillin-resistant, non-beta lactamase producing Enterococcus faeciumisolates from diverse geographic areas.” J. Clin. Microbiol. 30:2757-61). The vancomycin-resistant genotype (vanA, vanB or vanC) wasdefined by polymerase chain reaction analysis using specific primersselected from published gene sequences (Goering, R. V. and the MolecularEpidemiological Study Group (1994) “Guidelines for evaluating pulsedfield restriction fragment patterns in the epidemiological analysis ofnosocomial infections.” (Abstract) Third International Meeting ofBacterial Epidemiological Markers; Cambridge England).

Example 2 Isolation of VRE Phage

500 ml of raw sewage from the University of Maryland is mixed with 100ml of 10 times concentrated LB broth (Difco Laboratories). Thissewage-broth mixture is inoculated with a 18-24 hour LB broth culture (1ml) of a VRE strain and incubated at 37° C. for 24 hours to enrich themixture for bacteriophage which can infect the VRE strain added. Afterincubation, the mixture is centrifuged at 5000 g for 15 minutes toeliminate matter which may interfere with subsequent filtration. Thesupernatant is filtered through a 0.45 m Millipore filter. Filtrate isassayed using the Streak Plate Method and/or Appelman Tube TurbidityTest to detect lytic activity against different strains of VRE.

Method for Testing Phage Against VRE Isolates

Three methods are employed: Plaque Assay; Streak Plate Method; and TubeTurbidity Method, and the procedures for each follow.

Plaque Assay:

A 18-24 hour nutrient broth culture of the VRE strain (0.1 ml) to betested for susceptibility to infection and dilutions of a VRE phagepreparation (1.0 ml) arc Mixed and then added to 4.5 ml 0.7% molten agarin nutrient broth at 45° C. This mixture is completely poured into apetri dish containing 25 ml of nutrient broth solidified with 2% agar.During overnight incubation at 37° C., VRE grow in the agar and form aconfluent lawn with some VRE cells being infected with phage. Thesephages replicate and lyse the initially infected cells and subsequentlyinfect and lyse neighboring bacteria. However the agar limits thephysical spread of the phage throughout the plate, resulting in smallvisibly clear areas called plaques on the plate where bacteriophage hasdestroyed VRE within the confluent lawn of VRE growth.

The number of plaques formed from a given volume of a given dilution ofbacteriophage preparation is a reflection of the titer of thebacteriophage preparation. Also, since one plaque with a distinctmorphology represents one phage particle that replicated in VRE in thatarea of the bacterial lawn, the purity of a bacteriophage preparationcan be ensured by removing the material in that plaque with a pasteurpipette (a “plaque pick”) and using this material as the inoculum forfurther growth cycles of the phage. On this basis, doing further plaqueassays on preparations of phage grown from this plaque pick, one wouldexpect all plaques to have a single appearance or plaque morphologywhich is the same as the plaque picked, a further indication of purity.Therefore this technique can not only be used to test bacteriophagepotency but also bacteriophage purity.

Streak Plate Method:

Eighteen hour LB broth cultures of the different enterococci strains tobe tested are grown at 37° C. (resulting in approximately 10⁹ CFU/ml)and a loopful of each culture is streaked across a nutrient agar platein a single line. This results in each plate having a number ofdifferent VRE streaked across it in single straight lines of growth.Single drops of phage filtrates to be tested are applied to the steaksof each VRE growth, and the plate is incubated 6 hours at 37° C., atwhich time the steaks of the different VRE strains are examined for theability of phage to form clear areas devoid of bacterial growth,indicating lysis of that particular VRE strain by that particular phage.

The VRE host range for a given phage filtrate can be ascertained bywhich VRE streaks it is capable of causing a clear area devoid of growthand which strains of VRE the phage is incapable of doing this.

Appelman Tube Turbidity Test (from Adams, M. H. 1959. Bacteriophages.Interscience Publ. New York N.Y.):

18 hour LB broth cultures of different VRE strains are prepared. 0.1 mlof phage filtrate or a dilution thereof is added to 4.5 ml of VRE brothcultures and incubated at 37° C. for 4 hours (monophages) or 4-18 hours(polyvalent phages). Phage free VRE broth cultures are used as controls.Broth cultures which are normally turbid due to bacterial growth areexamined for the ability of the phage to lyse the VRE strain asindicated by the clearing of the culture turbidity.

The host range of a given phage can be ascertained by which VRE brothcultures the phage is capable of clearing and which broth cultures itcannot induce clearing.

Example 3 A Phage Strain is Active Against Over 200 VRE Isolates

A collection of 234 VRE isolates, 187, E. faecium of which 3 strains arefrom ATCC, 41 E. faecalis strains, and 6 E. gallinarium strains as wellas 6 E. faecium strains which are vancomycin sensitive were tested forsusceptibility of infection by 7 monophages isolated as described inExample 2. Susceptibility of infection was determined by the 3techniques described. The majority of VRE strains in this collectionwere isolated from patients at the University of Maryland and BaltimoreVA Medical Centers as indicated in Example 1. Such VRE isolates weredetermined to be distinct and genetically diverse by pulsed field gelelectrophoresis typing. Of the 7 monophages, VRE/E2 and VRE/E3 have arelatively narrow host range compared to other VRE phages, but are ableto infect the small proportion of VRE strains which were resistant toother phages collected. A phage cocktail containing the above 7 VREmonophages lysed 95% of the VRE strains in the collection.

Example 4 Producing Bacteriophage-Containing Compositions

0.1 ml amounts of a 18-24 LB broth culture¹ of a strain of VRE, whichhas been previously selected on the basis of being able to produce amaximum yield of bacteriophage are mixed with 1.0 ml of a VRE monophagefiltrate and then mixed with 4.5 ml of 0.7% molten agar in nutrientbroth at 450 C. T-his mixture is completely poured into a petri dishcontaining 25 ml of nutrient broth solidified with 2% agar. Afterovernight incubation at 37° C., the soft top agar layer with the phageis recovered by gently scraping it off the plate, and this recoveredlayer is mixed with a small volume of broth (1 ml per plate harvested).This suspension is centrifuged at 5,000-6,000 g for 20 minutes at 4° C.and the phage containing supernatant is carefully removed. Thesupernatant is filtered through a 0.45 m filter and centrifuged at30,000 g for 2-3 hours at 4° C. ¹LB broth culture contains Bacto LBBroth. Miller (Luria-Bertani, dehydragted) reconstituted according toinstructions by Difco Laboratories, Detroit, Mich.

The phage containing pellet is suspended in 1-5 ml of phosphate bufferand is further purified by ion exchange chromatography using a Qresource ion exchange column (Pharmacia Biotech Piscataway N.J.) and a0-1 M NaCl gradient in the start buffer. Phage tends to be eluted fromthe column between 150-170 mM NaCl with each fraction being assessed forthe presence of phage by standard plaque assay technique. Fractionscollected and assayed are pooled if the phage titer by the plaque assayis no greater than 3 logs lower than the phage preparation put onto thecolumn (e.g., 10¹⁰ PFU/ml is put onto the column therefore pool onlythose fractions with titers >10⁷ PFU/ml). Pooled fractions are testedfor endotoxin by the Limulus Amebocyte Lysate Assay (BioWhittaker IncWalkersville Md.). Pools demonstrating, >50 EU/ml of endotoxin arepassed through a Affi-prep polymyxin support column (Bio-Rad Labs,Hercules, Calif.) to remove residual endotoxin.

The phage pool is buffer exchanged against 100 mM ammonium bicarbonateusing size exclusion with Sephadex 0-25 chromatography (PharmaciaBiotech). 1 ml aliquots of the purified phage are freeze dried in thepresence of gelatin and stored at room temperature. The purity of thephage preparation is assessed by a combination of electron microscopy,SDS-PAGE, DNA restriction digest and analytical ultracentrifugation.

Example 5 Determination of a Protective Dose of Bacteriophage

Establishment of Sustained VRE Colonization in a Animal Model.

CD-1 mice are pretreated for seven days with 0.1 mg/ml of gentamicin and0.5 mg/ml of streptomycin in drinking water to reduce their normalintestinal flora.

VRE are then administered to the mice, who have fasted for 6 hours, byconsumption of one food pellet inoculated with 10⁶ CFU of VRE. VREintestinal colonization is confirmed in mice by standard colony countsof >10³ CFU VRE/gram of feces on CNA agar containing 10 g/ml ofvancomycin, 1 g/ml of amphotericin B and 10 g/ml of gentamicin. Thecolonization procedure is considered successful if there is consistentshedding of >10³ CFU of VRE per gram of feces for 5-7 days afterconsumption of the spiked food pellet. VRE colonization may persist for4 weeks by this method. Mice are given drinking water containing theabove mixture of antibiotics throughout the duration of the experiment.

Use of a in vivo mouse model to demonstrate efficacy of lyticbacteriophage in reducing VRE gastrointestinal colonization.

Twenty-four hours after detecting >10³ CFU VRE/grain of feces, mice wereadministered VRE phage (by having them consume one food pelletinoculated with 10⁹ PFU of VRE). Control groups consisted of (1)non-VRE-colonized mice sham dosed (no phage in dose), (2) VRE-colonizedmice which are sham dosed, and (3) non-VRE-colonized mice dosed withphage. Five mice were used in each group.

The efficacy of phage treatment to reduce VRE gastrointestinalcolonization was determined by quantitating VRE, on a daily basis, inweighed fecal samples from the mice in the different groups. Inaddition, at the end of the experiment, mice were sacrificed and thenumber of VRE and phage in their liver, spleen, and blood determined. Ifadministration of phage reduced VRE gastrointestinalcolonization/overall load in mice by at least 1 log as compared to thecontrol groups within 48-98 hours after phage administration, then thisdose of the particular phage was deemed efficacious. More preferably,colonization was reduced by at least 3 logs.

Example 6 Reduction of Colonization in Humans

The primary objective of this study is to (i) determine the efficacy ofa candidate phage preparation in transiently eliminating/reducing VREcolonization in humans, and (ii) further assess the kinetics of turnoverand the safety of the phages in immunocompromised patients, who are atgreatest risk for VRE infections. The study is a double-blinded,placebo-controlled trial of oral phage administration in hospitalizedpatients colonized with VRE.

VRE-colonized patients are enrolled in the study. The patients arerandomized to receive VRE-specific phages or a placebo. Stool samplesare collected immediately before administration of the phages or placeboand 1, 2 and 3 days after administration of the phages or placebo; forpatients who remain hospitalized, additional stool samples may beobtained 7 and 10 days after phage/placebo administration. The amount ofVRE and VRE-specific phages in the stools is quantitated, and data isrecorded on patient diagnosis, level of immunosuppression (as reflectedby the degree of neutropenia or administration of immunosuppressivemedications), and concurrent antibiotic therapy, if any. Side effects ofphage administration, and changes in blood counts and renal and liverfunction are noted.

Sufficient patients should be enrolled in each arm of the study toenable detection of a significant difference between groups (95%confidence, 80% power) if 20% of the group receiving phages are VREpositive 3 days after phage administration, vs. 50% of the groupreceiving a placebo. For these early Phase U efficacy studies, VRE casesare selected which are susceptible to the phage preparation in vitro; a“broad spectrum” VRE phage preparation may be tested during subsequent,more randomized clinical trials (i.e., phase III clinical trials). VREcounts will be compared before and after phage/placebo administration,in order to determine whether phage administration, even if noteradicating carriage, results in a significant (>1 log) decrease in VRElevels in stools. All VRE isolates will be screened for susceptibilityto the phage preparation. Most patients are expected to be colonizedwith only a single strain of VRE, some may have multiple strains;therefore, for a minimum of patients, 10-20 VRE colonies should bepicked from the primary isolation plate, in order to assess clonality(by PFGE) and for screening for phase susceptibility.

A successful outcome for the studies consists of the demonstration that(i) significantly more patients receiving phages became VREculture-negative than did patients receiving the placebo, or (ii) therewas a significantly greater decrease (>1 log) in VRE levels in thestools of persons receiving phages as compared with persons receivingthe placebo. From a clinical standpoint, there would be great value inreducing the levels of intestinal colonization during periods of severeneutropenia/immunosuppression, when the risk of bacteremia is greatest.

For purposes of charity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons of skill in medicine,bacteriology, infectious diseases, pharmacology, and/or related fieldsare intended to be within the scope of the invention, which is limitedonly by the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level .of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method for reducing the risk of bacterial infection or sepsis in asusceptible patient comprising treating the susceptible patient with apharmaceutical composition containing bacteriophage of one or morestrains which produce lytic infections in pathogenic bacteria.
 2. Themethod of claim 1, wherein treatment of the patient reduces the level ofcolonization with pathogenic bacteria susceptible to the bacteriophageby at least one log.
 3. The method of claim 1, wherein the susceptiblepatient is an immunocompromised patient selected from the groupconsisting of leukemia patients, lymphoma patients, carcinoma patients,sarcoma patients, allogeneic transplant patients, congenital or acquiredimmunodeficiency patients, cystic fibrosis patients, and AIDS patients.4. The method of claim 1, wherein the susceptible patient is colonizedwith the pathogenic bacteria subject to infection by said bacteriophage.5. The method of claim 1, wherein the pathogenic bacteria are selectedfrom vancomycin-resistant enterococcus (VRE), pneumococcal species,methicillin-resistant Staphylococcus aureus, multi-drug resistantStaphylococcus aureus (MDRSA), multi-drug resistant Pseudomonas species,Nesseria sp., Hemophilus sp., Proteus sp., Klebsiella sp. and Esherichiacoli.
 6. The method of claim 5, wherein the pathogenic bacteria areselected from VRE, MDSA, and multi-drug resistant Pseudomonas.
 7. Themethod of claim 1, wherein the bacteriophage composition is selectedfrom a parenteral composition, an oral tablet, capsule or liquid, anasal aerosol, a throat wash, a toothpaste, and a topical ointment. 8.The method of claim 1, wherein the patient has a wound selected from anulcer, a laceration, a deep penetrating wound and a surgical wound, thebacteriophage produce lytic infections in pathogenic bacteria capable ofinfecting these wounds.
 9. The method of claim 8, wherein thecomposition is a topical ointment, an irrigation solution or a componentof a wound dressing.
 10. The method of claim 1, wherein thepharmaceutical composition contains a plurality of bacteriophagestrains.
 11. The method of claim 10, wherein the pharmaceuticalcomposition contains bacteriophage strains which produce lyticinfections in pathogenic bacteria of a plurality of bacterial strains.12. The method of claim 10, wherein the pharmaceutical compositioncontains bacteriophage strains which produce lytic infections inpathogenic bacteria of a plurality of bacterial species.
 13. A methodfor reducing the incidence of infection by selected bacteria in amedical facility comprising administering a bacteriophage preparationwhich reduces the colonization level by the selected bacteria inpatients at risk for infection by the selected bacteria who are admittedto said medical facility.
 14. The method of claim 13, wherein thepatients at risk for infection are selected from the group consisting ofleukemia patients, lymphoma patients, carcinoma patients, sarcomapatients, allogeneic transplant patients, congenital or acquiredimmunodeficiency patients, cystic fibrosis patients, and AIDS patients.15. The method of claim 13, wherein said bacteriophage is administeredto substantially all patients admitted to said medical facility.
 16. Themethod of claim 13, wherein said bacteriophage is administered tosubstantially all patients colonized with the selected bacteria who areadmitted to said medical facility.
 17. (canceled)
 18. (canceled) 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. (canceled)24. A lytic bacteriophage which infects Enterococcus, wherein no morethan 30% of the Enterococcus strains in a collection of more than 100genetically diverse vancomycin resistant Enterococcus (VRE) strains areresistant to infection by said bacteriophage.
 25. (canceled) 26.(canceled)
 27. (canceled)
 28. (canceled)